Method of forming vertebrate pancreas in vitro

ABSTRACT

The present invention provides a pancreas induced in vitro that is useful for developmental engineering and organ engineering, and a method wherein a pancreas induced in vitro which contributes to the development of diagnosis and treatment of pancreatic disorders for higher animals, can artificially and efficiently be induced from a gastrula apart from the presumptive region of pancreas. An explant which has a secretory gland-like structure wherein several cells are gathered and which expresses insulin is formed in vitro by treating the blastopore upper lip of an early gastrula of a vertebrate such as  Xenopus  with retinoic acid in vitro, and then culturing. The treatment with retinoic acid can be carried out, for example, by treating with retinoic acid at a concentration of 10 −5  M or above for three hours.

TECHNICAL FIELD

The present invention relates to a method of forming pancreas in vitro,more particularly, a method of forming pancreas in vitro wherein ablastopore upper lip of the gastrula of a vertebrate is treated withretinoic acid and then cultured, and a screening method of a substanceeffective for the diagnosis and treatment of diseases attributed to apancreas induced in vitro and a pancreas using the pancreas induced invitro.

BACKGROUND ART

Every multicellular organism starts its development by fertilization andis completed as an individual having various tissues and well-balancedsystem by undergoing cell division (cleavage) and cell differentiation.The differentiation process is highly complicated and is thought thatimportant interactions between cells called induction phenomena takesplace in many steps of the differentiation stratum. The elucidation of“molecule that dominates morphogenesis” is said to be the mostsignificant. Amphibian embryos are often and mostly used as materialsfor these studies, nevertheless, the basic rule of body formation iscommon to all the vertebrates and homologous genes are known to havequite a similar function even among different species.

Amphibian embryo has conventionally been regarded as an extremelyvaluable material in the field of experimental embryology with whichmany studies have been made. This is because amphibian egg fertilizesand develops externally, its large egg makes embryo operation possible,and its time course changes can easily be observed. The amphibianblastopore upper lip of gastrula is a special region and when it istransplanted into the ventral side of another embryo, a secondary embryoincluding head or body-tail part is induced. This is why the blastoporeupper lip is named “organizer” as a region that acts as the center ofmorphogenesis that determines the embryo system. It is well known thatthe organizer induces central nerve by functioning to presumptiveectoderm during invagination of the primitive gut, while the organizeritself differentiates into dorsal mesoderm and anterior endoderm.

On the other hand, pancreas is an endocrine organ which indicateshistomorphology and manner of development that is common to mostvertebrates, namely, mammals, birds, reptiles and amphibians, and is anexocrine organ as well. It is known that during the developmentalprocess, dorsal and ventral primordia arise from the endoderm, and theyfuse to be pancreas (Development 121, 1569-1580, 1995).

It has been said that there exists a mesoderm in the vicinity ofendoderm in the process of embryogenesis, and that action frommesenchyme to endoderm is necessary for the differentiation of pancreas(Dev. Biol. 4, 242-255, 1962). Recent studies have reported thatinvolvement of notochord is required for pancreatic formation of chick,and that notochord suppresses the Shh expression in the endoderm in itsvicinity to differentiate pancreas. It has also been reported that it isthe endoderm of the pancreas presumptive region that differentiates intopancreas by action of notochord, and that differentiation into pancreasis not found in endoderm aside from the pancreas presumptive region,even when notochord coexists (Development 124, 4243-4252, 1997, Proc.Natl. Acad. Sci. USA 95, 13036-13041, 1998, Genes and Dev. 12,1705-1713, 1998).

Further, from research at gene level, it had been reported that homeoboxgene, known as ipf-1 and pdx-1 that express in the pancreatic primordiumof mouse, is essential to the formation process of pancreas. Genetargeting experiment of ipf-1 revealed that mouse embryo without thisgene was pancreas-defective (Nature 371, 606-609, 1994). However, theprimordium of pancreas was formed even when this gene was deficient, andthe existence of glucagon-positive cells was detected (Development 122,983-995, 1996). In addition, it is known that the vegetal pole cell ofXenopus blastula expresses both of XlHbox8 that is a pancreas-specifictranscription factor and IFABP that is the homolog of PDX-1 and a smallintestinal epithelium marker, however, when the signal of TGF-β systemat the endoderm is inhibited, the expression of XlHbox8 is inhibited(Development 122, 1007-1015, 1996).

On the other hand, it is known that retinoic acid is a regulatory factorfor the embryonic patterning along the anteroposterior axis (Nature 340,140-144, 1989, Development 112, 945-958, 1991, Dev. Biol. 192, 1-16,1997, Zool. Sci. 15, 879-886, 1998), and that this retinoic acidtransforms anterior neural tissue of Xenopus embryo to a posterior oneand is effective on mesodermal development (Genes Dev. 5, 175-187, 1991,Develop. Growth. Differ. 35, 123-128, 1993). It has also been reportedthat treatment with activin induces most mesodermal tissues such asnotochord, muscle, mesenchyme and coelomic epithelium, dose-dependentlyin Xenopus animal cap cells (Roux's Arch. Dev. Biol. 198, 330-335, 1990,Nature 347, 391-394, 1990, Roux's Arch. Dev. Biol. 200, 230-233, 1991).Changing the dosage of retinoic acid that is co-treated with activinenables the mesodermal tissues such as notochord, muscle and pronephrosthat differentiate from animal cap cells to be lateralized andposteriorized (Develop. Growth. Differ. 35, 123-128, 1993).

As to the action of retinoic acid to the endodermal organ, it has beenreported by Dixon et al. that when Xenopus embryos at developmentalstage 22 to 32 are treated with retinoic acid, the morphology of thedigestive organs such as the intestines, liver and stomach becomeabnormal, nevertheless, it has also been reported that pancreas ofXenopus embryos at developmental stage 22 to 32 that had been treatedwith retinoic acid is formed normally, and no effect is found in theexpression of XlHbox8, an endoderm-specific marker (Dev. Genes Evol.208, 318-326, 1998).

To date, specific induction of a specific organ in vitro had beenregarded as being extremely difficult, and the complex differentiationand formation mechanisms of pancreas remain unclear. The object of thepresent invention is to provide a method, whereby a pancreas induced invitro which enables to obtain findings on the differentiation andformation mechanisms of pancreas and thus is useful in developmentalengineering or organ engineering, a pancreas for transplantation bywhich it can be evaluated whether or not a pancreas induced in vitro canfunction in practice in vivo, and a pancreas induced in vitro whichcontributes to the development of diagnosis and treatment of pancreaticdiseases of higher animals, can be artificially and efficiently inducedfrom a gastrula excluding the presumptive region of pancreas.

DISCLOSURE OF THE INVENTION

The present inventors have conducted intensive study to elucidate theobject mentioned above, and have discovered that when the blastoporeupper lips of early gastrula, which, under normal conditions, do notform pancreas but form notochord and muscle, or pharnyx or the like whencultured in vitro, that is, the region to become dorsal mesoderm tissuesand anterior endodermal tissues, are cut out from the early gastrula ofXenopus and treated with retinoic acid, followed by culturing theseexplants in a Steinberg's solution containing BSA, their developmentalfate changed to pancreas, which can form morphologic and functionalpancreas at high efficiency in vitro. It was discovered that temporaryaction of retinoic acid is effective for the formation of pancreas bydifferetiation at high efficiency, and continuous treatment withretinoic acid does not induce differentiation of pancreas at highefficiency. Further, it was found out that 0 to 15 hours of precultureafter cutting out the blastopore upper lip, followed by retinoic acidtreatment is effective, which enables to induce differentiation ofpancreas at high efficiency. The present invention has been accomplishedbased upon these findings.

The present invention relates to: a method of forming vertebratepancreas in vitro, wherein a whole or a part of a vegetal pole sideregion of a vertebrate blastula or a gastrula is treated with retinoicacid in vitro, then cultured (claim 1); the method of forming vertebratepancreas in vitro according to claim 1, wherein the whole or the part ofthe vegetal pole side region of the blastula or the gastrula is a dorsalvegetal pole region of a blastula or a blastopore upper lip of agastrula (claim 2); the method of forming vertebrate pancreas in vitroaccording to claim 1 or 2, wherein the vertebrate is an animal thatbelongs to amphibian (claim 3); the method of forming vertebratepancreas in vitro according to claim 3, wherein the animal that belongsto amphibian is a Xenopus (claim 4); the method of forming vertebratepancreas in vitro according to any of claims 1 to 4, wherein an earlygastrula whose archenteron has not been formed yet is used as thegastrula (claim 5); the method of forming vertebrate pancreas in vitroaccording to any of claims 1 to 5, wherein a blastopore upper lip thatis cut out from the gastrula is used as the blastopore upper lip (claim6); the method of forming vertebrate pancreas in vitro according to anyof claims 1 to 6, wherein the treatment with retinoic acid is conductedto a blastopore upper lip that is precultured for 0 to 15 hours afterbeing cut out from the gastrula (claim 7); and the method of formingvertebrate pancreas in vitro according to any of claims 1 to 7, whereinthe treatment with retinoic acid is conducted by treating with retinoicacid at a concentration of 10⁻⁵ M or more for 0.5 to 5 hours (claim 8).

The present invention further relates to a pancreas induced in vitro,wherein said pancreas can be obtained by the method of formingvertebrate pancreas in vitro according to any of claims 1 to 8 (claim9), a screening method of a substance capable of curing the hypofunctionor dysfunction of pancreas wherein the pancreas induced in vitroaccording to claim 9 is used (claim 1), and a screening method of asubstance capable of detecting the hypofunction or dysfunction ofpancreas wherein the pancreas induced in vitro according to claim 9 isused (claim 11).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view showing the light-microscopic histology of a blastoporeupper lip that differentiates by treatment with retinoic acid for threehours.

FIG. 2 is a view showing the electron-microscopic histology of ablastopore upper lip that differentiates by treatment with retinoic acidfor three hours.

FIG. 3 is a view showing the change of gene expression of a blastoporeupper lip by treatment with retinoic acid for three hours.

BEST MODE OF CARRYING OUT THE INVENTION

There is no particular limitation to the method of forming vertebratepancreas in vitro in the present invention, as long as it is a methodwherein a whole or a part of a vegetal pole side region of a vertebrateblastula or a gastrula is treated with retinoic acid in vitro, thencultured, thereby enabling the differentiation and induction of pancreasin vitro. Aside from the pancreas organ induced in vitro, the pancreasin the method of forming vertebrate pancreas in vitro of the presentinvention includes the following, for convenience: an explant having anexpression ability of a pancreas-specific molecular marker gene, such asinsulin gene, homeobox gene such as ipf-1, pdx-1 or the like, XlHbox8gene that is a pancreas-specific transcription factor and the homolog ofpdx-1; an explant having a cytomorphology that is similar to pancreas invivo; and an explant having a secretory gland-like structure that issimilar to pancreas in vivo.

There is no particular limitation to the vertebrate mentioned above, aslong as it is an animal having a pancreas that belongs to mammal, bird,reptile and amphibian. Nevertheless, in a level where findings on thedifferentiation and formation mechanisms of pancreas that is useful indevelopmental engineering and organ engineering can be obtained, ananimal that belongs to amphibian, which is relatively easy to handle andabundant knowledge of which developmental engineering and organengineering have been obtained to the present, a Xenopus, specifically,can particularly be exemplified as a preferable vertebrate.

As the blastula mentioned above, a mid-blastula to late-blastula canpreferably be used, and a specific example of said mid-blastula tolate-blastula is that of a Xenopus at developmental stage 8 to 9.Furthermore, as the gastrula mentioned above, it is preferable to use anearly gastrula whose archenteron has not been formed yet, which is anembryo at the developmental stage following blastula in the developmentof metazoa, and is in the process where a monolayered wall (blastoderm)is transformed to two layers of internal and external walls (germlayer). A specific example of said early gastrula is a gastrula of aXenopus at developmental stage 10 to 11. The developmental stage of thisXenopus can be determined from the criterion as described previously(Nieuwkoop, P. D., Faber, J., 1956. Nomal Table of Xenopus laevis.North-Holland Pub. Co. Amsterdam.). It is preferable to use a dorsalvegetal pole region of a blastula or a blastopore upper lip of agastrula, as a whole or a part of the vegetal pole side region of theblastula or the gastrula mentioned above.

Anything can be used as the blastopore upper lip mentioned above, aslong as it contains a blastopore upper lip of a gastrula, favorably ofan early gastrula at least. Using a blastopore upper lip cut out from anearly gastrula or the like by conventional means is preferable, and itis preferable for the blastopore upper lip which had been cut out fromthe gastrula to be precultured within 15 hours, preferably within fivehours, followed by treatment with retinoic acid.

Furthermore, there is no particular limitation to the treatmentcondition with retinoic acid mentioned above, such as to treatmentconcentration and treatment time, as long as it is a treatment conditionwherein the blastopore upper lip of vertebrate gastrula is treated invitro, followed by culturing, thereby enabling differentiation andinduction of pancreas. For example, as to treatment concentration,treatment with retinoic acid at a concentration of 10⁻⁵ M or above,preferably 10⁻⁴ M to 10⁻³ M can be given as an example, and as totreatment time, treatment with retinoic acid for 0.5 to 24 hours,preferably 0.5 to 5 hours can be given as an example. Since retinoicacid is not water-soluble, it is preferable to use the acid by firstdissolving with ethanol, dimethylsulfoxide (DMSO) and the like, thendiluting with physiological saline.

The pancreas induced in vitro of the present invention is notparticularly limited as long as it can be obtained by the method offorming pancreas in vitro mentioned above. As referred to above, asidefrom the pancreas organ induced in vitro, an explant having anexpression ability of a pancreas-like molecular marker gene, an explanthaving a cytomorphology that is similar to pancreas in vivo, and anexplant having a secretory gland-like structure that is similar topancreas in vivo are also included. Furthermore, as to the screeningmethod of the present invention, there is no particular limitation aslong as it is a screening method of a substance such as gene, peptide,protein and the like that are useful for diagnosis, treatment and othersthat use said pancreas induced in vitro. Examples of such substance aresubstance capable of curing the hypofunction or dysfunction of pancreas,substance capable of detecting the hypofunction or dysfunction ofpancreas, and the like. Screening of a substance that enhances orsuppresses the function of pancreas can be conducted, for example, byinjecting a test substance into a pancreatic cell of the pancreasinduced in vitro obtained from the present invention and comparing theexpression ability of marker molecules such as insulin or the like tothat of controls.

The present invention will be explained below in more detail with theexamples, but the technical scope of the invention will not be limitedto these examples.

EXAMPLE 1 Regional Culture of Gastrula and Presumptive Region ofPancreas

The dorsal lymph sacs of male and female Xenopuses were each injectedwith 600 IU of hCG (human chorionic gonadotropin; Gestron; DenkaSeiyaku, Japan), and eggs were obtained by mating these Xenopuses. Theseearly gastrulas [developmental stage 10; the developmental stage wasdetermined from the criterion as described previously (Nieuwkoop, P. D.,Faber, J., 1956. Nomal table of Xenopus laevis (Daudin). North-HollandPub. Co. Amsterdam.)] were dejellied in a Steinberg's solution (SS:58.00 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO₃)₂, 0.83 mM MgSO₄, 3.00 mMHEPES and 100 mg/l kanamycin sulfate; pH 7.4) containing 4.5% cysteinehydrochloride (pH 7.8), and the vitelline membrane was removed by usinga pair of watchmaker's tweezers.

The vegetal pole side region of the Xenopus early gastrulas mentionedabove (developmental stage 10) were cut by the regions mentioned bellowand cultured. That is, the vegetal pole side region was cut out so asnot to include the equatorial portion of the Xenopus early gastrula, anda region including the whole (Whole) of its portion, each regiondissected into one third (Dorsal, Intermediate and Ventral) verticallyto the dorsoventral axis, each region dissected into one third (Left,Center and Right) parallel to the dorsoventral axis, and only the dorsalregion of the central portion (Center-Dorsal) dissected into one thirdparallel to the dorsoventral axis were cut out, respectively. Theseregions were cultured for ten days in Steinberg's solution containing 1mg/ml BSA (bovine serum albumin; A-7888, Sigma). These cultured regionswere fixed with Bouin's solution for three hours, and were dehydrated bythe use of ethanol and xylene. These regions were embedded withparaffin, and the pieces that had been cut into a thickness of 6 μmsections were stained with hematoxylin and eosin. The tissues thatdifferentiated were observed and tested by using a light microscope. Theresults are shown in Table 1.

TABLE 1 Region Inter- Center- Whole Dorsal mediate Ventral Left CenterRight dorsal Number of specimens 22 21 24 15 37 16 39 20 Atypical  0 (0) 0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) epidermis Epidermis 19(86) 21 (100) 13 (54) 14 (93) 31 (84) 15 (94) 36 (92) 20 (100) Cementgland  0 (0)  6 (29)  1 (4)  0 (0)  1 (3)  4 (25)  0 (0)  0 (0) Eye orits  0 (0) 13 (62)  2 (8)  0 (0)  7 (19)  9 (56)  0 (0)  0 (0) fragmentsEar vesicle  0 (0)  1 (5)  0 (0)  0 (0)  4 (11)  2 (13)  1 (3)  0 (0)Neural tissue  5 (23) 13 (62)  6 (25)  0 (0) 21 (57) 12 (75) 23 (59)  3(15) Notochord 15 (68) 17 (81)  2 (8)  0 (0) 26 (70) 14 (88) 26 (67) 15(75) Muscle 19 (86) 19 (90) 17 (71)  6 (40) 29 (78) 13 (81) 36 (92)  9(45) Pronephros  0 (0)  1 (5) 10 (42)  7 (47) 11 (30)  1 (6) 16 (41)  0(0) Mesenchyme 21 (95) 21 (100) 23 (96)  7 (47) 26 (97) 15 (94) 39 (100)20 (100) Coelomic 19 (86)  9 (43) 21 (88) 15 (100) 31 (84) 12 (75) 31(79)  7 (35) epithelium Hemocyte  4 (18)  0 (0)  4 (17) 15 (100)  2 (5) 5 (31)  1 (3)  0 (0) Cartilage 17 (77) 19 (90)  3 (13)  0 (0) 24 (65)13 (81) 13 (33) 12 (60) Pharyngeal 19 (86) 19 (90) 18 (75) 10 (67) 29(78) 12 (75) 36 (92) 10 (50) epithelium Intestinal 20 (91)  1 (5) 12(50)  4 (27) 19 (51)  5 (31) 20 (51)  0 (0) epithelium Pancreas  4 (18) 2 (10)  4 (17)  3 (20) 13 (35)  4 (25) 19 (49)  1 (5) Gastric gland 16(73)  2 (10) 11 (46)  6 (40) 19 (51)  6 (38) 24 (62)  0 (0) Yolk-richcell  2 (100) 10 (48) 18 (75)  8 (53) 22 (59) 13 (81) 26 (67)  7 (35)

The results shown above revealed that head structures such as eyes,cement gland and the like differentiated well from the parts includingmany dorsal regions (Dorsal and Center), however, formation of headstructure could not be found from the parts mostly composed of thedorsal region (Center-Dorsal), wherein the dorsal mesodermal tissuessuch as notochord, muscle and cartilage differentiated. Since thesetissues are formed also in the parts that include many dorsal regions(Dorsal and Center), it can be considered that the head structure issecondarily induced from the dorsal mesodermal tissues.

When dissected into one third (Dorsal, Intermediate and Ventral)vertically to the dorsoventral axis, a dorsal mesoderm such as notochordand muscle differentiated from the dorsal region, and ventral mesodermtissues such as coelomic epithelium and hemocyte differentiated from theventral region. It was found out that when dissected into one third(Left, Center and Right) parallel to the dorsoventral axis, the headstructure differentiated well from the central portion, and aside fromthe fact that the differentiation rate of the pronephros is low,differentiation of almost all of the mesodermal tissues from the dorsalto ventral is commonly found in the three regions. Furthermore, as tothe endodermal tissues, there was a tendency that the formation rate ofintestinal epithelium and gastric gland from the regions containingdorsal portions (Dorsal and Center) was lower compared to that of otherregions.

Fundamentally, it is thought that in the archenteron period, thepresumptive region to become a pancreas is the dorsal right and leftside region of embryo. It was verified from the culture experimentaccording to the region of endoderm in the present example that pancreasis formed at highest rate from the right and left side regions of embryowhich had been dissected into one third parallel to the dorsoventralaxis (Left and Right). Nevertheless, the formation rate of pancreas atthese regions was not 100%, but 40 to 50%. As just described, whendissected into one third parallel to the dorsoventral axis, pancreas wasformed from the right and left side regions at a rate of approximately50% at the maximum. To the contrary, when dissected into one thirdvertically to the dorsoventral axis (Dorsal, Intermediate and Ventral),the formation rate of pancreas of all regions were as low as less than20%, and the formation rate of pancreas at the whole (Whole) of thevegetal pole side region was also low (18%). Based on these findings, itwas suggested that the presumptive region of pancreas is at the rightand left side region of embryo, and in the process where the presumptiveregion of pancreas differentiates into pancreas, the suppression ofpancreas formation by coexistence of the most dorsal region, thenecessity of coexistence of the ventral region, and the necessity of allthe regions along the dorsoventral axis direction were suggested. Henryet al. have reported that dorsal vegetal pole cells at developmentalstage 8 to 9 express both of XlHbox8 which is a pancreas-liketranscription factor and IFABP which is a marker of a small intestinalepidermis, but ventral vegetal pole cells only express IFABP and doesnot express XlHbox8 (Development 122, 1007-1015, 1996), and it isunderstood from this report that the dorsal vegetal pole cellsdifferentiate into pancreas. From the above, although there is adifference in that the vegetal pole side region is dissected into tworegions of dorsal and ventral in the report mentioned above, whereas itis dissected into one third and then differentiated into pancreas in thepresent example, it is considered that the presumptive region ofpancreas from the blastula to the early gastrula period is the dorsalright and left side regions among the vegetal pole side regions.

EXAMPLE 2 Formation of Pancreas from a Blastopore Upper Lip by UsingRetinoic Acid

A 0.3 mm×0.3 mm sized blastopore upper lip (the part which is thepresumptive region of the dorsal mesoderm and anterior endoderm) was cutout from an early gastrula of a Xenopus (developmental stage 10), thencultured for ten days at 20.degree. C. in a Steinberg's solutioncontaining 1 mg/ml BSA, and the dorsal mesoderm such as notochord andmuscle, anterior endodermal tissues such as the pharyngeal epitheliumand the neural tissues differentiated [Table 2, FIGS. 1A and 1B].Furthermore, immediately after this region was cut out, it was treatedwith retinoic acid at various concentrations as shown in Table 2 forthree hours. In the same manner as in that of Example 1, it was culturedfor ten days at 20.degree. C. and compared with the case where notreatment of retinotic acid had been carried out. Table 2 shows theresults obtained from observing and examining the tissues thatdifferentiated by using a light microscope. Note that in FIGS. 1A and1B, “not” represents notochord, “mus” represents muscle, “mes”represents mesenchyme, “epi” represents epidermis and “pha” representspharyngeal epithelium, respectively.

TABLE 2 Retinoic acid [M] 0 0 10⁻⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ 10⁻⁴ 10⁻³ (DMSO = 1%)Number of specimens 27 20 32 35 35 35 12 10 Atypical  0 (0)  0 (0)  0(0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) epidermis Epidermis 22 (81) 20(100) 31 (97) 34 (97) 13 (37)  8 (23) 10 (83) 10 (100) Cement gland  0(0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) Eye or its  0 (0) 0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) fragments Ear vesicle 0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) Neural tissue 13(48)  2 (10)  9 (28) 15 (43)  1 (3)  0 (0)  0 (0)  3 (30) Notochord 24(89) 18 (90) 31 (97) 30 (86) 25 (71) 33 (94) 11 (92) 10 (100) Muscle 22(81) 17 (85) 24 (75) 20 (57)  4 (11)  7 (20)  0 (0)  3 (30) Pronephros 0 (0)  0 (0)  0 (0)  0 (0) 12 (34)  0 (0)  0 (0)  0 (0) Mesenchyme 23(85) 20 (100) 32 (100) 35 (100) 20 (57) 32 (91) 12 (100) 10 (100)Coelomic  1 (4)  3 (15)  5 (16) 12 (34)  5 (14)  5 (14)  6 (50)  1 (10)epithelium Hemocyte  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0(0) Cartilage  9 (33)  5 (25) 16 (50) 22 (63)  3 (9)  0 (0)  0 (0)  0(0) Pharyngeal 17 (63) 17 (85) 30 (94) 34 (97) 19 (54) 21 (60) 10 (83) 6 (60) epithelium Intestinal  0 (0)  0 (0)  1 (3)  4 (11) 33 (94) 34(97) 12 (100)  0 (0) epithelium Pancreas  0 (0)  2 (10)  0 (0)  1 (3) 15(43) 25 (71) 11 (92)  0 (0) Gastric gland  0 (0)  0 (0)  0 (0)  1 (3)  9(26)  0 (0)  0 (0)  0 (0) Yolk-rich cell 21 (78) 16 (80) 24 (75) 31 (89)30 (86) 29 (83) 11 (92)  9 (90)

These results showed that the differentiation rate of neural tissues,muscle and cartilage decreased as the concentration of retinoic acidincreased. On the other hand, the notochord differentiated at high ratein any condition, regardless of the concentration of retinoic acid.Formation of intestinal epithelium and pancreas were found when theconcentration of retinoic acid increased, and its differentiation rateincreased as the concentration of retinoic acid increased. Thedifferentiation rate of pancreas almost reached plateau at around 10⁻⁴M, where 70 to 90% of the explants out of the total specimens formedpancreas, and it was discovered that this rate is higher that the casewhen regions containing presumptive region of pancreas of a normalembryo are cultured (40 to 50%). Formation of pronephros and gastricgland were also confirmed when treated with retinoic acid at 10⁻⁵ M. Bythe use of this method, formation of pancreas in vitro can be reproducedat a sufficiently high rate compared to when a presumptive region ofpancreas of a normal embryo is used, and thus, its use as a test systemthat elucidates the differentiation and formation of pancreas can beexpected.

The sections of tissues that were treated with retinoic acid solution at10-4 M mentioned above and then cultured for ten days, were stained withhematoxylin and eosin, then its histology was observed using a lightmicroscope. It was discovered that when pancreas is formed with highefficiency, in most cases, it also accompanied with the formation ofnotochord and intestinal epithelium, where the pancreas and notochordwere covered by intestinal epithelium [FIGS. 1C and 1D; “pan” representspancreas, “int” represents intestinal epithelium and “not” representsnotochord, respectively]. Table 2 shows the result of countingintestinal epithelium, since the intestine is not in a tubular form ascan be observed in vivo and the intestinal lumen of an organism isformed to be at the outer side the explant. Since this intestinal lumenis at the outer side of the explant and includes notochord and pancreasin the explant, it can be said that these conform to the configurationin vivo. Several pyramidal cells of the pancreas observed at this pointgathered and contacted each other to show a secretory gland-likestructure having small space in the inner side of the cells. Many ofthese cells gathered and presented morphology same as that of pancreasobserved in vivo. It was discovered that since the nucleus of cells thatcomposed the secretory gland structure existed toward the bottom side,it was common with the cells of pancreas in vivo.

Furthermore, the explant mentioned above, wherein a blastopore upper lipwas treated with retinoic acid at 10⁻⁴ M and then cultured for ten daysat 20.degree. C. in Steinberg's solution containing BSA, was treated inthe manner as described below and was observed by using an electronmicroscope. The cultured explants as described above were fixed inbuffer I (3% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M cacodylate;pH 7.4) for one day. These explants that had been fixed were washed withthe buffer I, then fixed in buffer II (1% OsO₄ and 0.1 M cacodylate; pH7.4) for two hours. After washing with the buffer II, the explants weredehydrated with ethanol and acetone, then embedded in epoxy resin. Theseexplants that had been embedded were cut in very thin slices anddouble-stained with uranyl acetate and lead citrate, then observed byusing a transmission electron microscope (JEM-200CX; JOEL) (see FIG. 2).Consequently, it can be verified that a number of cells gather to have acavity (lumen) inside, and there exist many exocrine granules in thecells near the cavity (FIG. 2A), and aside from the exocrine glandstructure and exocrine granule, existence of endocrine granule was alsoverified in the explants (FIGS. 2B, C). The scale bars of FIGS. 2A, 2Band 2C each represent 1, 1, 0.1 μm, and “Lu” represents lumen, “N”represents nucleus, the arrow in FIG. 2A represents exocrine granule,the arrow in FIG. 2B represents endocrine granule, respectively.

Moreover, the pharnyx and intestine were both observed by the lightmicroscope mentioned above, as an endodermal epithelium that composesthe exodermis of the explants. They did not have a tubular form whereinthe cavity in the gastrointestinal tract is in the inner side as can beseen in vivo, and the cavity in the gastrointestinal tract is in theouter side of the explants. Further, the pharnyx and intestine are bothendodermal epithelia, but it is known that the height of the cells arelow and a form where cuboidal cells are lined is shown in a pharyngealepithelium, while the height of the cells are tall and the epithelium iscomposed of tall cells lined in an intestinal epithelium. Those with theheight/width ratio of cells lower than three were classified as thepharyngeal epithelium and those with the ratio higher than three werecounted as intestinal epithelium (Table 2). The tissues that wereclassified as intestinal epithelium here resembled to the morphology ofesophagus and duodenum of an organism. Since these endodermal epitheliaare connected from the mouth to the anus in vivo, their appearancechange gradually, and it is also difficult to classify minutely only bydetermination based on the shape of each part of the gastrointestinaltract that does not have a specific structure such as a gastric gland orthe like. Therefore, it had been decided here to classify to pharyngealepithelium and intestinal epithelium, according to the thickness of theendodermal epithelium. Observation by a microscope showed that as theconcentration of retinoic acid increased, the formation rate of theintestinal epithelium increased, while that of the pharyngeal epitheliumtended to decrease. However, since it was counted even if there was onlya little existing, regardless of the ratio found in the explant, thenumeric value of the differentiation rate of the pharyngeal epitheliumin a high concentration retinoic acid treatment is higher than theimpression by the actual observation.

Since retinoic acid powder is not water-soluble, it was used by firstdissolving with ethanol and DMSO, then diluted with Steinberg's solutioncontaining 1 mg/ml BSA. Therefore, in order to examine the influence ofDMSO on tissue differentiation, DMSO was added to Steinberg's solutioncontaining 1 mg/ml BSA to a final concentration of 1%, and a blastoporeupper lip was cultured for ten days at 20.degree. C. in the same manneras described above (Table 2). Consequently, from the verification thatformation of pancreas and intestinal epithelium are not induced only by1% DMSO, it has been discovered that this has no effect on tissuedifferentiation.

EXAMPLE 3 Expression of Pancreas Specific Genes

The expression of tissue-specific genes in an explant that had beentreated with retinoic acid was examined. A blastopore upper lip treatedwith retinoic acid at 10⁻⁴ M, or an untreated blastopore upper lip werecultured for three days, and their total RNA were extracted from each ofthe blastopore upper lip, and cDNA was synthesized by using a reversetranscriptase (GIBCO BRL). PCR reaction was conducted to 1 μl of thecDNA (2 μg/μl) obtained, and study and comparison were made between theexpression pattern of insulin which is a gene specific to pancreas(Proc. Natl. Acad. Sci. USA 88, 7679-7683, 1991) and XlHbox8 (homolog ofPDX1) (Dev. Biol. 171, 240-251, 1995), and the expression pattern ofNCAM (neural cell adhesion molecule) which is a neural marker gene and αskeletal muscle actin (muscle-specific actin: ms-actin) that is a musclemarker gene. EF-1α (elongation factor 1α) was used as a loading control.The combination of primers of each gene in PCR reaction that were usedis as follows:

insulin (Seq. ID No. 1) [insulin-F: 5′-ATGGCTCTATGGATGCAGTG-3′, (Seq. IDNo. 2)] insulin-R: 5′-AGAGAACATGTGCTGTGGCA-3′, X1Hbox8 (Seq. ID No. 3)[X1Hbox8-F: 5′-CCTACAGCAACCCCTTGGTA3′, (Seq. ID No. 4)] X1Hbox8-R:5′-GGGCTCTTGTGTAGGCTGTC-3′, NCAM (Seq. ID No. 5) [NCAM-F:5′-CACAGTTCCACCAAATGC-3′, (Seq. ID No. 6)] NCAM-R:5′-GGAATCAAGCGGTACAGA-3′, ms-actin (Seq. ID No. 7) [ms-actin-F:5′-AACAGCAGCTTCTTCCTCAT-3′, (Seq. ID No. 8)] ms-actin-R:5′-TACACAGAGCGACTTGAACA-3′, EF-1α (Seq. ID No. 9) [EF-1α-F:5′-TTGCCACACTGCTCACATTGCTTGC-3′, (Seq. ID No. 10)] EF-1α-R:5′-ATCCTGCTGCCTTCTTTTCCACTGC-3′.

76 μl of DDW, 10 μl of 10×Ex buffer, 8 μl of 2.5 mM dNTPs mix, 0.5 μl of5U/μl ExTaq, and 0.5 μl of 100 μM of each of the above-mentioned primerwere added to 5 μl of the above-mentioned cDNA (20 ng/μl), and PCRreaction was conducted with the total amount of 100 μl. The thermalcycle program, which is a cycle to denature for four minutes at94.degree. C. only at the first time, followed by thermal denaturationfor 30 seconds at 94.degree. C., stretched for one minute at 58.degree.C., and then annealing for one minute at 72.degree. C., was repeated for23 to 30 times. Ultimately, annealing was conducted for nine minutes at72.degree. C. After the PCR amplification product was isolated byagarose gel (1.5%) electrophoresis, it was detected by southernhybridization (see FIG. 3). A normal embryo (whole embryo) atdevelopmental stage 41 was used as a positive control.

It can be said from the result of FIG. 3 described above that NCAM andms-actin express well with untreated blastopore upper lip [dorsal lip(−RA)], and although a little expression could be found in insulin andXlHbox8, they were very weak. Meanwhile, the expression of NCAM andms-actin were suppressed while the expression of insulin and XlHbox8were strongly promoted in the explants treated with retinoic acid[dorsal lip+RA]. Based on these findings, it was revealed that neuronand muscle are formed from untreated blastopore upper lip but pancreasdo not differentiate, however, when treated with retinoic acid, theformation of neuron and muscle are suppressed, and differentiation ofpancreas is induced.

EXAMPLE 4 The Influence of Treatment Time of Retinoic Acid

Because the strength of drug treatment is effected by concentration andtime, first, in order to discuss pancreas formation only from the pointof retinoic acid concentration, the effect due to time was eliminated byculturing in a retinoic acid solution, the treatment time of theblastopore upper lip with retinoic acid as described in theabove-mentioned Example 2 was extended from three hours to 10 days, andthe effect on pancreas formation by treatment with retinoic acid at eachconcentration shown in Table 3 were examined. From these results, it wasrevealed that regarding the tissues aside from pancreas and pronephros,the differentiation manner of the tissues was almost the same as that ofthe explants that had been treated with retinoic acid for three hours.In other words, it was found out that because of the existence ofretinoic acid in the culture solution, the formation ratio of neuron,muscle and cartilage decrease, and the formation ratio of intestinalepithelium increase. However, as to pancreas and pronephros, tissuedifferentiation was not induced with high efficiency in any of theconcentration of retinoic acid treatment. Comparing this result with theresult when treated with retinoic acid for three hours, the formationrate of pancreas remained at about 10% in the same manner as that of theretinoic acid treatment of a concentration at 10⁻⁶ M and its vicinitywhen treated for three hours, and pancreas was not formed with highefficiency. Based on this result, it was considered that the formationof pancreas is suppressed when continuously exposed to retinoic acid.Furthermore, retinoic acid treatment at 10⁻⁴ M, which is theconcentration wherein pancreas is formed with high efficiency whentreated for three hours, was also conducted. However, at thisconcentration, all the explants died while being cultured.

TABLE 3 Retinoic acid [M] 0 10⁻⁹ 10⁻⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ Number of specimens16 11 20 15 16 17 Atypical  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)epidermis Epidermis 16 (100) 11 (100) 19 (95)  8 (53)  1 (6)  7 (41)Cement  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0 (0) gland Eye or its  0 (0) 0 (0)  0 (0)  0 (0)  0 (0)  0 (0) fragments Ear  0 (0)  0 (0)  0 (0)  0(0)  0 (0)  0 (0) vesicle Neural tissue  8 (50)  9 (82)  7 (35)  2 (13) 0 (0)  0 (0) Notochord 13 (81) 11 (100) 17 (85) 14 (93) 14 (88) 14 (82)Muscle 10 (63)  9 (82) 12 (60)  4 (27)  3 (19)  0 (0) Pro-  0 (0)  0 (0) 0 (0)  1 (7)  0 (0)  0 (0) nephros Mesen- 15 (94) 10 (91) 19 (95)  9(60) 13 (81)  8 (47) chyme Coelomic  3 (19)  5 (45)  6 (30)  9 (60)  2(13)  2 (12) epithelium Hemocyte  0 (0)  0 (0)  0 (0)  0 (0)  0 (0)  0(0) Cartilage  7 (44)  5 (45)  9 (45)  0 (0)  0 (0)  0 (0) Pharyn- 10(63)  7 (64) 12 (60)  9 (60)  3 (19)  5 (29) geal epithelium Intestinal 0 (0)  0 (0)  2 (10)  5 (33) 14 (88)  8 (47) epithelium Pancreas  0 (0) 0 (0)  1 (5)  1 (7)  1 (6)  2 (12) Gastric  0 (0)  0 (0)  0 (0)  3 (20) 2 (13)  0 (0) gland Yolk-rich  7 (44)  8 (73)  9 (45) 13 (87) 14 (88)15 (88) cell

In 1960, it had been reported by Okada that action by mesoderm isnecessary to the differentiation of endodermal organs (Roux's Arch 152,1-21, 1960). From this report, it can be considered that the suppressionof pancreas formation by continuous treatment of retinoic acid showsdirect effect on the endoderm and indirect effect on the mesoderm.However, since the differentiation manner of the mesodermal tissue showsalmost the same tendency when treated for three hours and when treatedcontinuously, it can be presumed that the possibility of indirectsuppressive action to the mesoderm is low. Further, in order to formpancreas with high efficiency from the blastopore upper lip, temporaryaction with retinoic acid is necessary, and it is considered thatexposing for a long period suppresses the formation of pancreas. First,in the process of pancreas formation in a normal development, thepresumptive regions of liver and pancreas are isolated from the regionsaround it by the formation of liver diverticulum, and a part of itdifferentiates into pancreas. It can be also considered from this factthat there is a possibility that continuous exposure to some signalneeds to be avoided also in the development of normal pancreas.

EXAMPLE 5 The Timing of the Treatment with Retinoic Acid

It was made clear from the Example mentioned above that the primarydevelopmental fate of the blastopore upper lip of early gastrula can bechanged by treatment with retinoic acid. Regarding the endodermal organ,in particular, since its formation in a normal development is slowercompared to that of organs derived from other germ layers, it can beexpected that the period when its differentiation is determined is alsoslow. It was found out that it is effective to the pancreas formation toconduct retinoic acid treatment immediately after cutting out from theearly gastrula, and then treating for three hours (Table 2). Then, bychanging the time lag between the cutting out of the blastopore upperlip and the treatment with retinoic acid, the effective period of actionof pancreas induction of retinoic acid was confirmed, and thedetermination time of endodermal organs in the blastopore upper lip wasestimated.

Blastopore upper lips were cut out from an early gastrula of a Xenopus,followed by preculture for 0, 5, 15 and 25 hours, and each of them weretreated with retinoic acid at 10⁻⁴ M. The treatment time of thisretinoic acid was set to one hour in order to narrowly analyze theeffect of the treatment timing, and after culturing for 10 days in aSteinberg's solution containing BSA, the tissues that differentiatedwere examined (Table 4). The results showed that pancreas was formedfrom the blastopore upper lip in those that were precultured 0 and 5hours after being cut out, and pancreas was not formed in thoseprecultured 15 and 25 hours. Regarding the other tissues, an intestinalepithelium was formed aside from notochord and mesenchyme in thoseprecultured for 0 and 5 hours. However, as to those treated withretinoic acid after preculture for more than 15 hours, the formation ofintestinal epithelium was suppressed and the differentiation rate ofepithelium, coelomic epithelium and pharyngeal epithelium increased.

TABLE 4 Retinoic acid [M] 10⁻⁴ Preculture time 0 5 15 25 Treatment time1 Number of specimens 34 36 40 34 Atypical  0 (0)  0 (0)  0 (0)  0 (0)epidermis Epidermis 11 (32)  5 (14) 13 (33) 28 (82) Cement gland  0 (0) 0 (0)  0 (0)  0 (0) Eye or its  0 (0)  0 (0)  0 (0)  0 (0) fragmentsEar vesicle  0 (0)  0 (0)  0 (0)  0 (0) Neural tissue  0 (0)  0 (0)  0(0)  0 (0) Notochord 33 (97) 36 (100) 39 (85) 33 (97) Muscle  1 (3)  0(0)  0 (0)  1 (3) Pronephros  0 (0)  0 (0)  0 (0)  0 (0) Mesenchyme 23(68) 30 (83) 40 (95) 34 (100) Coelomic  0 (0)  0 (0) 28 (30) 16 (47)epithelium Hemocyte  0 (0)  0 (0)  0 (0)  0 (0) Cartilage  0 (0)  0 (0) 0 (0)  0 (0) Pharyngeal 25 (74) 27 (75) 37 (60) 34 (100) epitheliumIntestinal 34 (100) 33 (92) 26 (10)  5 (15) epithelium Pancreas 24 (71)19 (53)  0 (5)  0 (0) Gastric gland  0 (0)  0 (0)  8 (0)  0 (0)Yolk-rich cell 32 (94) 30 (83) 29 (45) 20 (59)

It was revealed from the above that the effect of retinoic acid on theformation of pancreas is limited to the blastopore upper lipsimmediately after being cut, those that were precultured for 5 hours,and those that were precultures for 15 hours, and the action of retinoicacid lost its effect in case the preculture was conducted for more than15 hours. It can be considered that the disappearance of responseability of the blastopore upper lip to the retinoic acid and thedetermination of developmental fate of endodermal organs occur in thisperiod. Further, it was presumed that those precultured for 5 to 15hours from the gastrula period belong to developmental stage 12 to 18,and that the developmental fate of the endodermal organs is notdetermined at this period even at the normal developmental stage. Infact, it can be considered that the period wherein the endodermal organsare formed (determined) at normal development and in vitro almostcoincide, since the liver diverticulum is formed and the differentiationand formation of endodermal organs such as liver and pancreas startduring the developmental stage 13 at normal development. It is expectedalso from this point that the present test system can be usedeffectively in research related to the formation of pancreas.

INDUSTRIAL APPLICABILITY

Although the activity of pancreas such as insulin is vital to adultdiseases such as diabetes and the like, the complex mechanisms ofdifferentiation and formation of pancreas have not been understood tothe present. According to the present invention, since pancreas can beproduced with high efficiency in vitro, it not only enables to obtainknowledge regarding the mechanisms of differentiation and formation ofpancreas that is useful for developmental engineering and organengineering, but also contributes to the development of diagnosis andtreatment of pancreas disorder of higher animals.

1. A method of forming an explant that expresses insulin in vitro,comprising: (a) isolating a Spemann-Mangold organizer region from avertebrate gastrula; (b) culturing said Spemann-Mangold organizer regionfor a predetermined period of time, wherein the period is determined bya comparison of pancreatic differentiation levels of saidSpemann-Mangold organizer region which is cultured for different periodsof time and treated with retinoic acid; (c) treating saidSpemann-Mangold organizer region with retinoic acid of a predeterminedconcentration for a predetermined period of time, wherein theconcentration is determined by a comparison of the pancreaticdifferentiation levels of said Spemann-Mangold organizer region whichare treated with retinoic acid of different concentrations, and theperiod is determined by a comparison of pancreatic differentiationlevels of said Spemann-Mangold organizer region which is treated withretinoic acid; and (d) culturing said Spemann-Mangold organizer regionto produce an explant that expresses insulin.
 2. The method according toclaim 1, wherein the vertebrate is an amphibian.
 3. The method accordingto claim 2, wherein the amphibian is a Xenopus.
 4. The method accordingto claim 1, wherein the gastrula is an early gastrula prior to formationof an archenteron.
 5. The method according to claim 1, wherein thctreatment with retinoic acid is conducted to a Spemann-Mangold organizerthat is cultured for 0 to 15 hours after being cut out from thegastrula.
 6. The method according to claim 1, wherein the treatment withretinoic acid is conducted by treating with retinoic acid at aconcentration of at least 10⁻⁵ M for 0.5 to 5 hours.